Nicotine dependence, in the form of tobacco smoking, is a major source of preventable morbidity and mortality worldwide. Despite the currently available therapeutic interventions, successful smoking quit rates remain low. Thus, there is an urgent need to develop new and more efficacious treatments. Accumulating evidence suggests a crucial role of excitatory glutamate transmission in the mesocorticolimbic system in mediating nicotine reinforcement, suggesting that glutamate receptors may be innovative targets for the treatment of nicotine addiction. Decreasing glutamate transmission by activation of the presynaptic inhibitory metabotropic glutamate receptor 2/3 (mGluR2/3) attenuates nicotine taking and seeking. However the relative contribution of each receptor (2 vs 3) in nicotine dependence is unknown due to the lack of subtype selective pharmacological tools. Further, little is known regarding the role of the inhibitory presynaptic mGluR7 in nicotine dependence. The only commercially available mGluR7 agonist, AMN082, is far from optimal because of the off-target effects produced by its metabolite. This project seeks to develop the molecular tools that will enable the cell-specific overexpression of these three mGluR subtypes in specific brain sites to allow us to investigate the role of each one of these receptors in nicotine dependence. Lentivirus-mediated genetic manipulations permit the efficient transfer of genes into the brain and sustain long-term regulation of the target receptor(s). This targeted experimental approach allows one to probe the mechanistic basis of nicotine dependence. Work under Specific Aim 1 will generate and validate viral vectors to overexpress mGluR2, 3 and 7 on glutamate terminals in the ventral tegmental area (VTA), a brain site within the mesolimbic system that is critically involved in nicotine dependence and where there is high density of these receptors. Neuron non-specific viral vectors will be generated and validated in vitro to examine the function mediated by vector-expressed mGluRs. Glutamatergic neuron-specific vectors will also be generated. All the vectors will be validated in vivo to examine vector-induced expressions and changes of protein and mRNA levels in the rat brain. Specific Aim 2 will initiate work towards the investigation of the role of each mGluR in the reinforcing and motivational effects of nicotine by using these molecular tools. Specifically, viral vectors will be injected ino the VTA, after which nicotine self-administration will be conducted. We hypothesize that overexpression of mGluR2, 3 or 7 will attenuate nicotine self-administration. In summary, this project will provide novel molecular tools, and then initiate the application of these tools in the investigation of the involvement of mGluR subtypes in nicotine dependence using well-established behavioral rat procedures. Findings from this work will provide the foundation for a future R01 NIH grant application that will systematically explore the role of these mGluRs in different aspects of nicotine dependence.